The present invention relates to pharmaceutical compositions containing, as active principle, a 4,5-dihydro-1,3-thiazol-2-ylamine derivative of formula (I): 
or to one of the pharmaceutically acceptable salts thereof, to the novel derivatives of formula (I) and to their preparation.
The compounds of formula (I) are inhibitors of nitric oxide synthase and particularly of the inducible isoform of this enzyme.
Nitric oxide (NO) is a diffusable radical involved in many physiological and pathological processes. It is synthesized by oxidation of L-arginine, this reaction being catalyzed by a family of enzymes known as nitric oxide synthases or NO-synthases (NOSs), which is referenced in the international enzyme nomenclature system under the number E.C. 1.14.13.39.
Three NOS isoforms, two of which are constitutive and one inducible, are known:
a neuronal NOS (NOS-1 or nNOS) was originally isolated and cloned from nerve tissue in which it is a constitutive enzyme. NOS-1 produces NO in response to various physiological stimuli such as the activation of membrane receptors according to a mechanism dependent on calcium and on calmodulin;
an inducible NOS (NOS-2 or iNOS) can be induced in response to immunological stimuli such as, for example, cytokines or bacterial antigens in various cells such as, for example, macrophages, endothelial cells, hepatocytes, glial cells, as well as many other types of cell. The activity of this isoform is not regulated by calcium. Consequently, once induced, it produces large amounts of NO over prolonged periods.
an endothelial NOS (NOS-3 or eNOS) is constitutive and calcium/calmodulin-dependent. It was originally identified in vascular endothelial cells, in which it generates NO in response to physiological stimuli such as the activation of membrane receptors.
The NO produced by the neuronal and endothelial constitutive isoforms (NOS-1 and NOS-3) is generally involved in intercellular signalling functions. For example, the endothelial cells which line the inner wall of the blood vessels induce the relaxation of the underlying smooth muscle cells via the production of NO. It thus contributes towards regulating the arterial pressure.
The NO produced in large amount by the inducible isoform NOS-2 is, inter alia, involved in pathological phenomena associated with acute and chronic inflammatory processes in a large variety of tissues and organs.
An excessive production of NO by induction of NOS-2 thus plays a part in degenerative pathologies of the nervous system such as, for example, multiple sclerosis, cerebral, focal or global ischemia, cerebral or spinal trauma, Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, migraine, depression, schizophrenia, anxiety and epilepsy. Similarly, aside from the central nervous system, the induction of NOS-2 is involved in numerous pathologies with inflammatory components, such as, for example, diabetes, atherosclerosis, myocarditis, arthritis, arthrosis, asthma, irritable bowel syndrome, Crohn""s disease, peritonitis, gastro-esophageal reflux, uveitis, Guillain-Barrxc3xa9 syndrome, glomerulonephritis, lupus erythematosus and psoriasis. NOS-2 has also been implicated in the growth of certain forms of tumors such as, for example, epitheliomas, adenocarcinomas or sarcomas, and in infections with Gram-positive or Gram-negative intracellular or extracellular bacteria.
In all the situations in which an overproduction of NO is deleterious, it thus appears to be desirable to reduce the production of NO by administering substances capable of inhibiting NOS-2.
Thiazoline-based NOS inhibitors are described in particular in patent applications WO 94/12165, WO95/11231 and WO 96/14842.
The pharmaceutical compositions according to the present invention are those containing, as active principle, a derivative of formula (I) in which R represents an -alk-S-alk-Ar radical, a phenyl radical or a phenyl radical substituted with alkoxy or halogen, Ar is a phenyl radical and alk represents an alkylene radical.
When R is a substituted phenyl it is preferably monosubstituted, and in particular in position 3 or 4.
In the preceding definitions and in those which follow, the alkyl, alkylene and alkoxy radicals and the alkyl and alkoxy portions contain 1 to 6 carbon atoms in a straight or branched chain.
The halogen atoms are bromine, chlorine, iodine and fluorine atoms, and more particularly the bromine atom.
The alkoxy radicals are, in particular, methoxy, ethoxy and propoxy radicals, and more preferably methoxy radicals
R preferably represents a phenyl radical, a phenyl radical which is monosubstituted with alkoxy and more particularly with methoxy or a halogen atom and more particularly with a bromine atom.
The compounds of formula (I) contain one or more asymmetric carbon atoms and can thus be in racemic form or in the form of enantiomers and diastereoisomers; these also form part of the invention, as well as mixtures thereof.
Moreover, the compounds of formula (I) can be in the tautomeric form (Ia): 
These tautomers also form part of the invention.
The preferred pharmaceutical compositions are those containing a compound of formula (I), the racemic mixture, enantiomers and diastereoisomers thereof, the tautomer thereof and the pharmaceutically acceptable salts thereof chosen from the following compounds: 4-(3-bromophenyl)-4,5-dihydro-1,3-thiazol-2-ylamine 4-(4-methoxyphenyl)-4,5-dihydro-1,3-thiazol-2-ylamine 4-phenyl-4,5-dihydro-1,3-thiazol-2-ylamine 4-(benzylsulfanylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine.
The pharmaceutical compositions that are even more preferred are those containing, as active principle, a compound of formula (I), the tautomer thereof or pharmaceutically acceptable salts thereof, chosen from the following compounds:
(4RS)-4-(3-bromophenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
4-(4-methoxyphenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
(xe2x88x92)-(4R)-4-(4-methoxyphenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
(xe2x88x92)-(4R)-4-phenyl-4,5-dihydro-1,3-thiazol-2-ylamine.
The derivative of formula (I) for which R is phenyl is known (Chem. Abst., registry Number 76999-87-6).
The other derivatives of formula (I) are novel and as such form part of the invention.
The compounds of formula (I) that are preferred are the following compounds:
4-(3-bromophenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
4-(4-methoxyphenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
4-(benzylsulfanylmethyl)-4,5-dihydro-1,3-thiazol-2-ylamine
the racemic mixtures, enantiomers, diastereoisomers and mixtures thereof, the tautomers thereof and the pharmaceutically acceptable salts thereof.
The compounds that are even more preferred are the following:
(4RS)-4-(3-bromophenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
4-(4-methoxyphenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
(xe2x88x92)-(4R)-4-(4-methoxyphenyl)-4,5-dihydro-1,3-thiazol-2-ylamine
the tautomers thereof and the pharmaceutically acceptable salts thereof.
The compounds of formula (I) can be prepared by cyclization of a derivative of formula: 
in which R has the same meanings as in formula (I).
This cyclization is generally carried out using an acid such as hydrochloric acid, in aqueous medium, at a temperature of 100xc2x0 C. 6N hydrochloric acid is preferably used.
The derivatives of formula (II) can be obtained according to the reaction scheme below: 
In these formulae, R has the same meanings as in formula (I), Ra represents a hydrogen atom or an alkyl or alkoxycarbonyl radical, preferably methyl, ethyl or isobutyloxycarbonyl, and Rb is a hydrogen atom or a protecting group for the amine function such as those described by T. W. Greene, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), and preferably an acetyl or tert-butoxycarbonyl radical.
The reduction step a is preferably carried out using a hydride such as sodium borohydride or lithium aluminum hydride, in a (1-4C) aliphatic alcohol or tetrahydrofuran, at a temperature of between 10xc2x0 C. and 30xc2x0 C., or alternatively using a borane derivative such as the BH3-THF complex, in a solvent such as tetrahydrofuran, at a temperature of between 0xc2x0 C. and 30xc2x0 C.
The deprotection reaction b for the compounds for which Rb is a protecting group for the amine function is carried out by any deprotection method known to those skilled in the art, and in particular those described by T. W. Greene, Protective groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). When the protecting group is an acetyl radical, this reaction is preferably carried out using aqueous hydrochloric acid, at a temperature of 100xc2x0 C. When the protecting group is a tert-butoxycarbonyl radical, this reaction is carried out using hydrochloric acid in dioxane, at a temperature in the region of 20xc2x0 C.
Reaction c is carried out by the action of tert-butyl isothiocyanate, in an inert solvent such as a (1-4C) aliphatic alcohol (preferably methanol or ethanol), in the presence of a tertiary amine such as triethylamine, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The intermediates A are commercially available or can be prepared by application or adaptation of the methods described in the examples, and in particular by the following methods:
When R is phenyl substituted with halogen, the intermediate A can be obtained from the corresponding halobenzaldehyde by the action of potassium hydroxide and aqueous ammonia, in the presence of lithium chloride and benzyltriethylammonium chloride, in a solvent such as a mixture of dichloromethane, chloroform and water at a temperature of between 0xc2x0 C. and 30xc2x0 C., optionally followed by an esterification by the action of a (1-4C) aliphatic alcohol (preferably methanol or ethanol), in the presence of an inorganic acid such as sulfuric acid, at a temperature of between 50xc2x0 C. and the boiling point of the reaction medium. When R is phenyl substituted with alkoxy, Ra is an alkyl radical and Rb is tert-butoxycarbonyl, the intermediate A can be obtained by alkylation of the corresponding N-tert-butoxycarbonylhydroxyphenylglycine by the action of an alkyl halide (for example methyl iodide), in the presence of a base such as potassium carbonate, in an inert solvent such as dimethylformamide, at a temperature of between 0xc2x0 C. and 30xc2x0 C.
The compounds of formula (I) for which R is a radical -alk(1C)-S-alk-Ar may also be prepared by the action of a derivative of formula: 
in which X is a halogen atom and preferably iodine, or a tosyl radical, Ra and Rb are hydrogen atoms or protecting groups for the amine function such as those described by T. W. Greene, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably alkoxycarbonyl or acetyl and more particularly tert-butoxycarbonyl, with a derivative of formula HS-alk-Ar in which Ar represents a phenyl radical and alk is an alkylene radical (1-6C in a straight or branched chain), followed, if necessary, by a deprotection of the amine function.
This reaction is generally carried out in the presence of a base such as potassium carbonate, in a solvent such as acetonitrile or dimethylformamide and preferably acetonitrile, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The deprotection reaction for the compounds for which Ra or Rb is a protecting group for the amine function is carried out by any deprotection method known to those skilled in the art and in particular those described by T. W. Greene, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). Preferably, when the protecting group is an acetyl radical, this reaction is carried out using aqueous hydrochloric acid, at a temperature of 100xc2x0 C. When the protecting group is a tert-butoxycarbonyl radical, this reaction is carried out using hydrochloric acid in dioxane, at a temperature in the region of 20xc2x0 C.
The compounds of formula (III) may themselves be obtained according to the following reaction scheme: 
In these formulae, Ra and Rb are a hydrogen atom or a protecting group for the amine function such as those described by T. W. Greene, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991), preferably alkoxycarbonyl or acetyl and more particularly tert-butoxycarbonyl, and Ts is a tosyl radical.
Reaction a is generally carried out by the action of tert-butyl isothiocyanate, in an inert solvent such as an aliphatic (1-4C) alcohol (preferably methanol or ethanol), optionally in the presence of a tertiary amine such as triethylamine, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
Cyclization reaction b is generally carried out using an acid such as hydrochloric acid, in aqueous medium, at a temperature in the region of 100xc2x0 C. 6N hydrochloric acid is preferably used.
When Ra or Rb is a tert-butoxycarbonyl group, reactions c and g are carried out by any protection method known to those skilled in the art and in particular those described by T. W. Greene, Protective Groups in Organic Synthesis, J. Wiley-Interscience Publication (1991). This reaction is preferably carried out using di-tert-butyl dicarbonate, in the presence of a base such as triethylamine and optionally in the presence of 4-(dimethylamino)pyridine, in a solvent such as dichloromethane and at a temperature in the region of 20xc2x0 C., or alternatively in the presence of a base such as potassium carbonate, in a solvent such as water and at a temperature in the region of 20xc2x0 C.
Reaction d is generally carried out by the action of p-toluenesulfonyl chloride, in the presence of a tertiary amine such as triethylamine, in an inert solvent such as dichloromethane, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
Reaction e is generally carried out by the action of sodium iodide, in an inert solvent such as acetone, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
Reaction f is generally carried out by the action of an allyl halide, for example allyl chloride, in an aliphatic (1-4C) alcohol, preferably ethanol, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
Reaction h is generally carried out by the action of iodine, in the presence of a base such as sodium bicarbonate, in a solvent such as dichloromethane, at a temperature of between 20xc2x0 C. and the boiling point of the reaction medium.
The compounds of formula (I) are isolated and can be purified by the usual known methods, for example by crystallization, chromatography or extraction.
The enantiomers of the compounds of formula (I) can be obtained by resolving the racemic mixtures, for example by chromatography on a chiral column according to Pirckle W.H. et al., Asymmetric Synthesis, Vol. 1, Academic Press (1983) or by formation of salts or by synthesis from chiral precursors. The diastereoisomers can be prepared according to the known conventional methods (crystallization or chromatography or from chiral precursors).
The compounds of formula (I) can optionally be converted into addition salts with an inorganic or organic acid by the action of such an acid in an organic solvent such as an alcohol, a ketone, an ether or a chlorinated solvent. These salts also form part of the invention.
Examples of pharmaceutically acceptable salts which may be mentioned are the following salts: benzenesulfonate, hydrobromide, hydrochloride, citrate, ethanesulfonate, fumarate, gluconate, iodate, isethionate, maleate, methanesulfonate, methylenebis-b-oxynaphthoate, nitrate, oxalate, pamoate, phosphate, salicylate, succinate, sulfate, tartrate, theophyllineacetate and p-toluenesulfonate.
The compounds of formula (I) are inhibitors of inducible NO-synthase or type-2 NO-synthase (NOS-2) and are thus useful for preventing and treating disorders associated with excessive NO production, such as multiple sclerosis, cerebral, focal or global ischemia, cerebral or spinal trauma, Parkinson""s disease, Huntington""s disease, Alzheimer""s disease, amyotrophic lateral sclerosis, migraine, depression, schizophrenia, anxiety and epilepsy, diabetes, atherosclerosis, myocarditis, arthritis, arthrosis, asthma, irritable bowel syndrome, Crohn""s disease, peritonitis, gastro-esophageal reflux, uveitis, Guillain-Barre syndrome, glomerulonephritis, lupus erythematosus, psoriasis, the growth of certain forms of tumors such as, for example, epitheliomas, adenocarcinomas or sarcomas, and infections with Gram-positive or Gram-negative intracellular or extracellular bacteria.
Their activity as NOS-2 inhibitors was determined by measuring the conversion of [3H]-L-arginine into [3H]-L-citrulline with, respectively, an NOS-2 enzymatic fraction extracted from the lungs of rats or mice pretreated with lipopolysaccharides (10 mg/kg i.p. 6 hours before collecting the tissue). The compounds were incubated for 20 to 30 minutes at 37xc2x0 C. in the presence of 5 xcexcM of [3H]-L-arginine, 1 mM of NADPH, 15 xcexcM of tetrabiopterine, 1 xcexcM of FAD and 0.1 mM of DTT in a HEPES buffer (50 mM, pH 6.7) containing 10 xcexcg/ml of calmodulin. The incubation was stopped by adding cold HEPES buffer (100 mM, pH 5.5) containing 10 mM of EGTA and 500 mg of a cationic ion-exchange resin (AG50W-X8, counterion: Na+) to separate the [3H]-L-arginine from the [3H]-L-citrulline. After separation of the phases by settling for 5 min, the radioactivity remaining in the liquid phase was measured in a scintillation counter in the presence of a suitable scintillation liquid. The yield for the recovery of the [3H]-L-citrulline formed was able to be estimated using [14C-ureido]-L-citrulline as external standard.
The activity was expressed as picomole(s) of [3H]-L-citrulline formed per minute and per milligram of protein contained in the reaction medium.
In this test, the IC50 value of the compounds of formula (I) is less than or equal to 1 xcexcM.
The compounds of formula (I) are of low toxicity. Their LD50 value is greater than 40 mg/kg via the subcutaneous route in mice.